The knotted1 (kn1) gene, originally isolated from maize by transposon tagging, encodes a nuclear homeodomain-containing transcription factor active in a regulatory network controlling the meristematic state of cells, which in turn regulates cell development and differentiation. Hake, et al., (1989) EMBO J. 8:15-22; Volbrecht, et al., (1991) Nature 350:241-243; Sinha, et al., 1993 Genes and Development 7:787-795. Knotted1 is the founding member of a family of homeodomain proteins conserved in higher plants. Homologues of knotted1 with conserved homeodomains have been isolated from a variety of species, including rice (Matsuoka, et al., (1993) Plant Cell 5:1039-1048), Arabidopsis (Ruberti, et al., (1991) EMBO J. 10:1787-1791; Mattsson, et al., (1992) Plant Mol. Bio. 18:1019-1022; Schena and David, (1992) PNAS 89:3894-3898; Lincoln, et al., (1994) Plant Cell 6:1859-1876), soybean (Ma, et al., (1994) Plant Molecular Biology 24:465-473), barley (Muller, et al., (1995) Nature 374:727-730), sorghum (Malcomber, et al., GenBank DQ317417) and wheat (Ishida and Takumi, GenBank AB465042). This class of proteins is characterized by a conservation of amino acid residues in the recognition helix and N-terminus of the homeodomain; further sequence homologies among kn1-related genes are found in the 24 amino acids immediately upstream of the homeodomain, referred to as the ELK region. Kerstetter, et al., (1994) The Plant Cell 6:1877-1887; Burglin, (1997) Nucleic Acids Research 25(21):4173-4180; Burglin, (1998) Dev. Genes. Evol. 108:113-116.
Tobacco plants expressing maize Kn1 under a strong constitutive promoter displayed a range of altered phenotypes generally including malformed leaves, shortened internodes, loss of apical dominance and the formation of epiphyllic shoots. Sinha, et al., (1993) Genes and Development 7:787-795. Phenotypic similarity between cytokinin-overproducing Arabidopsis and Kn1-overexpressing plants suggested that a single pathway is involved and that cytokinins may act upstream of kn1, inducing its expression. Rupp, et al., (1999) Plant Journal 18(5):557-563. However, expression of maize kn1 in tobacco, under control of the senescence-associated SAG12 promoter, delayed leaf senescence and increased leaf cytokinin content by as much as 15-fold. Ori, et al., (1999) Plant Cell 11:1073-1080. Thus, kn1 expression and cytokinin levels may positively regulate each other in a complex interdependency. D'Agostino and Kieber, (1999) Current Opinions in Plant Biology 2:359-364. The KN1 homeodomain proteins may play pivotal roles in maintaining leaf cells in an indeterminate state. Immunolocalization studies have demonstrated that the KN1 protein is nuclear and thus consistent with the predicted function of kn1 gene as a transcription factor.
The KN1-type homeodomain proteins have been subdivided into two groups, classes 1 and 2 (Kerstetter, et al., 1994). Class 1 includes the maize kn1 gene. The class 1 products share extensive amino acid identity in the homeodomain and in general, they are strongly expressed around the shoot meristem, moderately to weakly expressed in the embryo and/or other restricted tissues and barely expressed in differentiated organs, such as leaves and roots (Kerstetter, et al., 1994). Ectopic expression of kn1-like class 1 genes has been reported to cause altered leaf and flower morphology in spontaneous mutants of a number of plant species (Liu, et al., (2008) Journal of Genetics and Genomics 35:441-449; Smith, et al., (1992) Development 116:21-30; Chen, et al., (1997); Parnis, et al., (1997)) and in transgenic plants (Matsuoka, et al., (1993) Plant Cell 5:1039-1048; Lincoln, et al., (1994) Plant Cell 6:1859-1876). The class 2 genes, which are comparatively less similar to maize kn1 in their homeodomains, are expressed in most tissues at different levels, depending upon the tissue. In contrast to the class 1 genes, overexpression of class 2 genes in transgenic plants does not cause altered morphology.
Cytokinins are a class of N6 substituted purine derivative plant hormones that regulate cell division and influence a large number of developmental events, such as shoot development, sink strength, root branching, control of apical dominance in the shoot, leaf development, chloroplast development and leaf senescence (Mok, et al., (1994) Cytokinins. Chemistry, Action and Function. CRC Press, Boca Raton, FLA, pp. 155-166; Horgan, (1984) Advanced Plant Physiology ed. MB., Pitman, London, UK, pp 53-75 and Letham, (1994) Annual Review of Plant Physiol 34:163-197). In maize, cytokinins (CK) play an important role in establishing seed size, decreasing tip kernel abortion and increasing seed set during unfavorable environmental conditions (Cheikh, et al., (1994) Plant Physiol. 106:45-51; Dietrich, et al., (1995) Plant Physiol Biochem 33:327-36). Active cytokinin pools are regulated by rates of synthesis and degradation.
Until recently, roots were believed to be the major site of cytokinin biosynthesis but evidence indicates that others tissues, such as shoot meristems and developing seeds, also have high cytokinin biosynthetic activity. It has been suggested that cytokinins are synthesized in restricted sites where cell proliferation is active. The presence of several Atipt genes in Arabidopsis and their differential pattern of expression might serve this purpose.
The enzyme isopentenyl transferase (IPT) directs the synthesis of cytokinins and plays a major role in controlling cytokinin levels in plant tissues. Multiple routes have been proposed for cytokinin biosynthesis. Transfer RNA degradation has been suggested to be a source of cytokinin, because some tRNA molecules contain an isopentenyladenosine (iPA) residue at the site adjacent to the anticodon (Swaminathan, et al., (1977) Biochemistry 16:1355-1360). The modification is catalyzed by tRNA isopentenyl transferase (tRNA IPT; EC 2.5.1.8), which has been identified in various organisms such as Escherichia coli, Saccharomyces cerevisiae, Lactobacillus acidophilus, Homo sapiens and Zea mays (Bartz, et al., (1972) Biochemie 54:31-39; Kline, et al., (1969) Biochemistry 8:4361-4371; Holtz, et al., (1975) Hoppe-Seyler's Z Physiol. Chem. 356:1459-1464; Golovko, et al., (2000) Gene 258:85-93 and Holtz, et al., (1979) Hoppe-Seyler's Z Physiol. Chem. 359:89-101). However, this pathway is not considered to be the main route for cytokinin synthesis (Chen, et al., (1997) Physiol. Plant 101:665-673 and McGraw, et al., (1995) Plant Hormones, Physiology, Biochemistry and Molecular Biology Ed. Davies, 98-117, Kluwer Academic Publishers, Dordrecht).
Another possible route of cytokinin formation is de novo biosynthesis of iPMP by adenylate isopentenyl transferase (IPT; EC 2.5.1.27) with dimethylallyl-diphosphate (DMAPP), AMP, ATP and ADP as substrates. Current knowledge of cytokinin biosynthesis in plants is largely deduced from studies on a possible analogous system in Agrobacterium tumefaciens. Cells of A. tumefaciens are able to infect certain plant species by inducing tumor formation in host plant tissues (Van Montagu, et al., (1982) Curr Top Microbiol Immunol 96:237-254; Hansen, et al., (1999). Curr Top Microbiol Immunol 240:21-57). To do so, the A. tumefaciens cells synthesize and secrete cytokinins which mediate the transformation of normal host plant tissues into tumors or calli. This process is facilitated by the A. tumefaciens tumor-inducing plasmid which contains genes encoding the necessary enzyme and regulators for cytokinin biosynthesis. Biochemical and genetic studies revealed that Gene 4 of the tumor-inducing plasmid encodes an isopentenyl transferase (IPT), which converts AMP and DMAPP into isopentenyladenosine-5′-monophosphate (iPMP), the active form of cytokinins (Akiyoshi, et al., (1984) Proc. Natl. Acad. Sci. USA 81:5994-5998). Overexpression of the Agrobacterium ipt gene in a variety of transgenic plants has been shown to cause an increased level of cytokinins and elicit typical cytokinin responses in the host plant (Hansen, et al., (1999) Curr Top Microbiol Immunol 240:21-57). Therefore, it has been postulated that plant cells use machinery similar to that of A. tumefaciens cells for cytokinin biosynthesis. Homologs of ipt have recently been identified in Arabidopsis and Petunia hybrida (Takei, et al., (2001) J. Biol. Chem. 276:26405-26410 and Kakimoto, (2001) Plant Cell Physiol. 42:677-685). Overexpression of the Arabidopsis ipt homologs in plants elevated cytokinin levels and elicited typical cytokinin responses in planta and under tissue culture conditions (Kakimoto, (2001) Plant Cell Physiol. 42:677-685).
Arabidopsis ipt genes are members of a small multigene family of nine different genes, two of which code for tRNA isopentenyl transferases and seven of which encode a gene product with a cytokinin biosynthetic function. Biochemical analysis of the recombinant AtIPT4 protein showed that, in contrast to the bacterial enzyme, the Arabidopsis enzyme uses ATP as a substrate instead of AMP. Another plant ipt gene (Sho) was identified in Petunia hybrida using an activation tagging strategy (Zubko, et al., (2002) The Plant Journal 29:797-808).
Regarding cytokinin biosynthesis and effect, see, for example, Ahikari, et al., (2005) Science 309:741-745; Cho, et al., (2002) Plant Growth Reg 36(3):215-221; Dietrich, et al., (1995) Plant Physiol. Biochem 33(3):327-336; Kaminek, (1992) Trends Biotech 10:159-164; Kokobun and Honda, (2000) Plant Prod. Sci. 3:354-359; Nagel, et al., (2001) Annals Bot. 88(1):27-31; Yashima, et al., (2005) Plant Prod. Sci. 8(2):139-144.
In view of the influence of cytokinins on a wide variety of plant developmental processes, including root architecture, shoot and leaf development and seed set, the ability to manipulate cytokinin levels in higher plant cells, and thereby drastically effect plant growth and productivity, offers significant commercial value (Mok, et al., (1994) Cytokinins. Chemistry, Action and Function. CRC Press, Boca Raton, Fla., pp. 155-166). The modulation of cytokinin, however, due to the many effects it has on plants and the multiple pathways for regulation and synthesis, is a complex process requiring careful temporal and spatial regulation in transgenic plants.
As can be seen, a continuing need exists for methods of modulation and characterization of developmental pathways for positively affecting crop plant yield.